Characterization of a novel glycine-rich protein from the cell wall of maize silk tissues

The isolation, characterization and regulation of expression of a maize silk-specific gene is described. zmgrp5 (Zea mays glycine-rich protein 5) encodes a 187 amino acid glycine-rich protein that displays developmentally regulated silk-specific expression. Northern, Western, in situ mRNA hybridization and transient gene expression analyses indicate that zmgrp5 is expressed in silk hair and in cells of the vascular bundle and pollen tube transmitting tissue elements. The protein is secreted into the extracellular matrix and is localized in the cell wall fraction mainly through interactions mediated by covalent disulphide bridges. Taken together, these results suggest that the protein may play a role in maintaining silk structure during development. This is the first documented isolation of a stigma-specific gene from maize, an important agronomic member of the Poaceae family.Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accessions nos. AY095207, AY095208 and AAM16282.1).     

A tomato cDNA inducible by salt stress and abscisic acid: nucleotide sequence and expression pattern

We have characterized a new tomato cDNA, TAS14, inducible by salt stress and abscisic acid (ABA). Its nucleotide sequence predicts an open reading frame coding for a highly hydrophilic and glycine-rich (23.8%) protein of 130 amino acids. Southern blot analysis of tomato DNA suggests that there is one TAS14 structural gene per haploid genome. TAS14 mRNA accumulates in tomato seedlings upon treatment with NaCl, ABA or mannitol. It is also induced in roots, stems and leaves of hydroponically grown tomato plants treated with NaCl or ABA. TAS14 mRNA is not induced by other stress conditions such as cold and wounding. The sequence of the predicted TAS14 protein shows four structural domains similar to the rice RAB21, cotton LEA D11 and barley and maize dehydrin genes.     

cDNA encoding a wheat (Triticum aestivum cv. Chinese Spring) glycine-rich RNA-binding protein

A wheat cDNA encoding a glycine-rich RNA-binding protein, whGRP-1, was isolated. WhGRP-1 contains two conserved domains, the RNA-binding motif (RNP motif) combined with a series of glycine-rich imperfect repeats, characteristic of a conserved family of plant RNA-binding proteins. Northern analysis revealed that whGRP-1 mRNA accumulates to high levels in roots and to lower levels in leaves of wheat seedlings. whGRP-1 mRNA accumulation is not enhanced by exogenous abscisic acid in seedlings and accumulates to very high levels during wheat embryo development, showing a pattern different from that of the ABA-inducible wheat Em gene.     

The Zea mays glycine-rich RNA-binding protein MA16 is bound to a ribonucleotide(s) by a stable linkage

Expression of the gene encoding the maize glycine-rich RNA-binding protein MA16 is developmentally regulated and it is involved in environmental stress responses. The MA16 protein shows a wide spectrum of RNA-binding activities. On the basis of in vivo labelling, where a [32P]phosphate label was linked to the MA16 protein, Freire and Pages (Plant Mol Biol 29:797-807, 1995) suggested that the protein may be post-translationally modified by phosphorylation. However, further analysis showed that the [32P]phosphate label was sensitive to different treatments, suggesting that modification distinct from protein phosphorylation might occur in the MA16 protein. Biochemical analysis revealed that this [32P]phosphate labelling was resistant to phenol extraction and denaturing SDS-PAGE but sensitive to micrococcal nuclease, RNase A and RNase T1 treatments. The mobility of [3?S] labelled MA16 protein on SDS-PAGE did not significantly changed after the nuclease treatments suggesting that the [32P]phosphate label associated to MA16 protein could be a ribonucleotide or a very short ribonucleotide chain. In addition, immunoprecipitation of labelled extracts showed that the ribonucleotide(s) linked to the MA16 protein was removed by phosphorolytic activity. This activity could be catalysed by a phosphate-dependent ribonuclease. The C-terminus of MA16 protein harbouring a glycine-rich domain was predicted to be an intrinsically disordered region.     

A maize glycine-rich protein is synthesized in the lateral root cap and accumulates in the mucilage.

The root cap functions in the perception of gravity, the protection of the root apical meristem, and facilitation of the passage of roots through the soil, but the genes involved in these functions are poorly understood. Here we report the isolation of a root-specific gene from the cap of maize (Zea mays L.) primary root by cDNA subtraction and differential screening. The gene zmGRP4 (Z. mays glycine rich protein 4) encodes a member of the glycine-rich proteins with a putative signal peptide at the amino terminus. The deduced molecular mass of mature zmGRP4 is 14.4 kD. In situ-hybridization analysis has shown zmGRP4 to be strongly expressed in the lateral root cap and weakly expressed in the root epidermis. A polyclonal antibody raised against recombinant zmGRP4 detected a protein of 36 kD in the insoluble protein fraction extracted from the root tip and the root proper, indicating posttranslational modification(s) of zmGRP4. Immunohistochemical analysis showed the accumulation of zmGRP4 in the mucilage that covers the root tip. These results indicate that lateral root-cap cells secrete modified zmGRP4 into the mucilage to which the protein may contribute to its characteristic physical properties.     

Structure and expression of the lignin O-methyltransferase gene from Zea mays L.

The isolation and characterization of cDNA and homologous genomic clones encoding the lignin O-methyltransferase (OMT) from maize is reported. The cDNA clone has been isolated by differential screening of maize root cDNA library. Southern analysis indicates that a single gene codes for this protein. The genomic sequence contains a single 916 bp intron. The deduced protein sequence from DNA shares significant homology with the recently reported lignin-bispecific caffeic acid/5-hydroxyferulic OMTs from alfalfa and aspen. It also shares homology with OMTs from bovine pineal glands and a purple non-sulfur photosynthetic bacterium. The mRNA of this gene is present at different levels in distinct organs of the plant with the highest accumulation detected in the elongation zone of roots. Bacterial extracts from clones containing the maize OMT cDNA show an activity in methylation of caffeic acid to ferulic acid comparable to that existing in the plant extracts. These results indicate that the described gene encodes the caffeic acid 3-O-methyltransferase (COMT) involved in the lignin biosynthesis of maize.     

AtGRP5, a vacuole-located glycine-rich protein involved in cell elongation

Although several glycine-rich protein (GRP) genes were isolated and characterized, very little is known about their function. The primary structure of AtGRP5 from Arabidopsis thaliana has a signal peptide followed by a region with high glycine content. In this work, green fluorescent protein fusions were obtained in order to characterize the sub-cellular localization of the AtGRP5 protein. The results indicated that this protein is the first described vacuolar GRP. Sense, antisense and RNAi transgenic A. thaliana plants were generated and analyzed phenotypically. Plants overexpressing AtGRP5 showed longer roots and an enhanced elongation of the inflorescence axis, while antisense and RNAi plants demonstrated the opposite phenotype. The analysis of a knockout T-DNA line corroborates the phenotypes obtained with the antisense and RNAi plants. Altogether, these results suggest that this vacuolar GRP could be involved in organ growth by promoting cell elongation processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Amanda Mangeon and Claudia Magioli contributed equally to this work.     

Organ-specific invertase deficiency in the primary root of an inbred maize line

An organ-specific invertase deficiency affecting only the primary root system is described in the Oh 43 inbred maize (Zea mays). Invertases (acid and neutral/soluble and insoluble) were assayed in various tissues of hybrid (NK 508) and inbred (Oh 43, W 22) maize lines to determine the basis for an early report that Oh 43 root tips were unable to grow on sucrose agar (27). Substantial acid invertase activity (7.3 to 16.1 micromoles of glucose per milligram of protein per hour) was evident in extracts of all tissues tested except the primary root system of Oh 43. This deficiency was also evident in lateral roots arising from the primary root. In contrast, morphologically identical lateral roots from the adventitious root system had normal invertase levels. These results suggest that ontogenetic origin of root tissues is an important determinant of invertase expression in maize. Adventitious roots (including the seminals) arise above the scutellar node and are, therefore, of shoot origin. The Oh 43 deficiency also demonstrated that invertase activity was not essential for maize root growth. Sucrose synthase was active in extracts from all root apices and theoretically provided the only available avenue for sucrose degradation in primary root tips of Oh 43. The deficiency described here will provide a useful avenue of investigation into the expression and significance of root invertase.     

Occurrence of Plant-Uncoupling Mitochondrial Protein (PUMP) in Diverse Organs and Tissues of Several Plants

The presence of plant-uncoupling mitochondrial protein (PUMP), previously described by Vercesi et al. (1995), was screened in mitochondria of various organs or tissues of several plant species. This was done functionally, by monitoring purine nucleotide-sensitive linoleic acid-induced uncoupling, or by Western blots. The following findings were established: (1) PUMP was found in most of the higher plants tested; (2) since ATP inhibition of linoleic acid-induced membrane potential decrease varied, PUMP content might differ in different plant tissues, as observed with mitochondria from maize roots, maize seeds, spinach leaves, wheat shoots, carrot roots, cauliflower, broccoli, maize shoots, turnip root, and potato calli. Western blots also indicated PUMP presence in oat shoots, carnation petals, onion bulbs, red beet root, green cabbage, and Sedum leaves. (3) PUMP was not detected in mushrooms. We conclude that PUMP is likely present in the mitochondria of organs and tissues of all higher plants.     

Amino acid sequence microheterogeneities of a type I cytokeratin of Mr 51,000 from Xenopus laevis epidermis

The complete cDNA-derived sequence of a type I cytokeratin (designated no. 3) from Xenopus laevis skin is described. The deduced protein has an Mr of 51,888 and consists of a glycine-rich head domain, a well-conserved alpha-helical region and a tail rich in hydroxyamino acid residues. Various cDNA clones encoding two different mRNAs were isolated that differed by short deletions/insertions and point mutations. These microheterogeneities are mainly located in a 'hypervariable region' at the C-terminal non-alpha-helical region.     

Gene sequence,developmental expression,and protein phosphorylation of RAB-17 in maize

The ABA-induced MA12 cDNA from maize, which encodes a set of highly phosphorylated embryo proteins, was used to isolate the corresponding genomic clone. This gene, called RAB-17 (responsive to ABA), encodes a basic, glycine-rich protein (mol. wt. 17 164) containing a cluster of 8 serine residues, seven of them contiguous. It is a homologue of the rice RAB-21 gene (Mundy J, Chua NH, EMBO J 7; 2279–2286, 1988). Phosphoamino acid analysis of the isolated protein indicates that only the serine residues are phosphorylated and a putative casein-type kinase phosphorylatable sequence was identified in the protein. The pattern of expression and in vivo phosphorylation of the RAB-17 protein was studied during maize embryo germination and in calli of both meristematic or embryonic origin. ABA treatment induced the synthesis of RAB-17 mRNA and protein in calli, however, the RAB-17 proteins were found to be highly phosphorylated only in embryos.